Stabilized hematoxylin

ABSTRACT

A stabilized hematoxylin composition is disclosed that includes one or both of a host compound and an antioxidant. The disclosed composition exhibits sufficient stability to be utilized in an automated staining process without undue degradation prior to use of the composition to stain a biological sample. Methods of using and making the stabilized composition also are disclosed.

RELATED APPLICATION DATA

This claims the benefit of U.S. Provisional Patent Application No.60/895,007, filed Mar. 15, 2007, which application is incorporated byreference herein.

FIELD

The present invention relates to a composition and method forhistochemical staining of biological samples. More particularly, thepresent invention relates to a dye formulation that is stabilizedagainst degradation over time, and use of the formulation to stainbiological samples.

BACKGROUND

Several histochemical staining protocols, including Hematoxylin andEosin (H&E) staining and Papanicolaou (PAP) staining, rely on the dyehematoxylin to stain cytological and tissue samples. In particular,hematoxylin staining of cell nuclei is used by pathologists to detectthe presence of malignant and/or metastatic cells in a tumor biopsysample.

Hematoxylin is a naturally-occurring compound found in the red heartwoodof trees of the genus Hematoxylon. Hematoxylin itself is colorless inaqueous solution and is not the active ingredient that stains tissuecomponents. Rather, an oxidation product of hematoxylin, hematein,becomes the active staining component of a hematoxylin dye solution,particularly upon complexation with a mordant. Hematein is producednaturally through exposure to air and sunlight. The natural process istermed “ripening,” and can take 3 or more months to provide a solutionsuitable for staining cells.

In order to accelerate the conversion of hematoxylin to hematein, achemical oxidant can be utilized. Unfortunately, the accelerated processoften produces ineffective reaction products such as oxyhematein andcomplex polymeric precipitates, and also provides a solution thatdegrades faster than a naturally ripened dye solution. The exact amountof oxidant needed to quantitatively oxidize hematoxylin to hematein canbe used to help avoid over-oxidation to ineffective products, but apartially-oxidized solution is more typically utilized when staining isnot performed immediately. In a partially-oxidized solution, naturaloxidation of the hematoxylin that is remaining after a chemicaloxidation step will continue to replace any hematein that is eitherconsumed during staining or is naturally oxidized further to ineffectiveproducts. Still, the concentration (and amount) of hematein can changeover time.

Since hematein is the active staining component of a hematoxylinsolution, changes in its concentration (and/or the concentration of itsmordant complexes) over time leads to staining inconsistencies. In amanual staining procedure, changes in hematein content of a hematoxylinsolution can be compensated for by altering the contact time of abiological sample with the solution based on visual inspection. Forexample, an apparently under-stained sample can simply be placed backinto the hematoxylin solution for a period of time to increase thestaining intensity. In an automated staining procedure, however,“visual” inspection and extension of the exposure time in response tounder-staining can require costly imaging equipment and can disruptprocessing of other samples. Therefore, a need exists for a hematoxylinsolution wherein the concentration of hematein available for staining isbetter stabilized over time.

SUMMARY

In one aspect, a stabilized hematoxylin composition is disclosed. In oneembodiment, the composition includes: a solvent; hematoxylin; an amountof a chemical oxidant sufficient to convert at least a portion of thehematoxylin to hematein; a mordant; and either or both of a hostcompound and an antioxidant. In a particular embodiment, a disclosedhematoxylin solution includes hematoxylin, water, a polyol, an amount ofan oxidant sufficient to convert at least a portion of the hematoxylinto hematein, a mordant, and either or both of an antioxidant and a hostcompound.

In another aspect, a method is disclosed for histochemical staining of abiological sample. The method includes contacting the biological samplewith a disclosed hematoxylin composition, and can further includecontacting the sample with one or more additional staining compositions,such as one or more counter-stains. In a particular embodiment, themethod further includes contacting the sample with an eosin composition.In another particular embodiment, the method is automated.

Also disclosed is a method for making a stabilized hematoxylincomposition that can be used for histochemical staining of a biologicalsample. In one embodiment, the method includes forming a hemateinsolution, adding a mordant to the hematein solution to form a stainingsolution, and adding either or both of a host compound and anantioxidant to the staining solution to form the stabilized hematoxylincomposition. The hematein solution can be formed by dissolving hemateindirectly in a solvent, by dissolving hematoxylin in a solvent and thenadding a chemical oxidant to convert at least a portion of thehematoxylin into hematein, or by a combination of dissolution ofhematein and conversion of hematoxylin to hematein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram outlining an automated H&E staining protocolinto which the disclosed hematoxylin composition can be incorporated.

FIG. 2 is diagram showing stability results for several embodiments ofthe disclosed hematoxylin composition.

FIG. 3 is another diagram showing stability results for severalembodiments of the disclosed hematoxylin composition.

DETAILED DESCRIPTION OF SEVERAL ILLUSTRATIVE EMBODIMENTS

The following description of several embodiments describes non-limitingexamples that further illustrate the invention. All titles of sectionscontained herein, including those appearing above, are not to beconstrued as limitations on the invention, but rather are provided tostructure the illustrative description of the invention that is providedby the specification. In order to aid the reader in understanding thevarious illustrated embodiments, explanations of particular termsutilized in the description are provided, after which an overview ofparticular embodiments of the invention and specific examples areprovided.

I. Terms:

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one skilled in the artto which the disclosed invention pertains.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly indicates otherwise. Thus, for example, reference to“a host compound” refers to one or more host compounds, such as 2 ormore host compounds, 3 or more host compounds, or even 4 or more hostcompounds.

The term “antioxidant” refers to an atom or molecule that has a greateroxidation potential than a second atom or molecule, such that theantioxidant is preferentially oxidized instead of the second atom ormolecule. For example, an antioxidant can have a greater oxidationpotential than hematein, and thus help prevent oxidation of hematein tooxyhematein. Furthermore, an antioxidant also can function as a reducingagent, for example, a reducing agent that converts oxyhematein back tohematein. Antioxidants can be present in the disclosed compositions atconcentrations ranging from about about 1 mM to about 1M, for example,from about 5 mM to about 500 mM, such as from about 50 mM to about 150mM.

The term “aqueous solvent” refers to a composition having water as themajor component and that is a liquid at room temperature. Mixtures ofwater and one or more lower alkanols or polyols that have 50% or greaterwater content by volume are examples of aqueous solvents.

The term “biological sample” refers to any sample that is obtained fromor otherwise derived from a biological entity such as an animal, forexample, a sample obtained from a human or a veterinary animal such as adog, cat, horse or cow. Examples of biological samples include cytologysamples, tissue samples and biological fluids. Non-limiting particularexamples of biological samples include blood, urine, pre-ejaculate,nipple aspirates, semen, milk, sputum, mucus, pleural fluid, pelvicfluid, sinovial fluid, ascites fluid, body cavity washes, eye brushings,skin scrapings, a buccal swab, a vaginal swab, a pap smear, a rectalswab, an aspirate, a needle biopsy, a section of tissue obtained forexample by surgery or autopsy, plasma, serum, spinal fluid, lymph fluid,sweat, tears, saliva, tumors, organs and samples obtained from in vitrocell or tissue cultures. Typically, the sample will be a biopsy samplethat has been fixed, processed to remove water and embedded in paraffinor another suitable waxy substance for cutting into tissue sections.Biological samples can be mounted on substrates such as microscopeslides for treatment and/or examination.

The term “hematoxylin composition,” as used herein, generically refersboth to compositions formed by dissolving hematein (the oxidationproduct of hematoxylin) directly into a solvent and to compositionsformed by dissolving hematoxylin in a solvent and allowing or promotingoxidation of the hematoxylin to hematein. Although it is more typical toprepare the disclosed compositions by dissolving hematoxylin in asolvent and converting the hematoxylin to hematein (either completely orpartially) by natural oxidation through contact with air or acceleratedchemical oxidation, the benefits of the stabilizing effects of thedisclosed composition components can also be utilized in combinationwith hematein compositions prepared by directly dissolving hematein insolvent. Thus, in some embodiments, a “hematoxylin composition” willinclude, at least initially, little or no hematoxylin, and consistprimarily of hematein.

The term “host compound” refers to an organic or inorganic molecule,complex or material having an inner cavity portion or groove portion,and more particularly, to a molecule having an inner cavity portion orgroove portion that can accommodate at least a portion of a hematein orother dye molecule. Host compounds include polysaccharides such asamyloses, cyclodextrins and other cyclic or helical compounds containinga plurality of aldose rings, for example, compounds formed through 1,4and 1,6 bonding of monosaccharides (such as glucose, fructose, andgalactose) and disaccharides (such as saccharose, maltose, and lactose).Other host compounds include cryptands, cryptophanes, cavitands, crownethers, dendrimers, nanotubes, calixarenes, valinomycins, andnigericins. In particular embodiments, a host compound can be acyclodextrin or cyclodextrin derivative, and more particularly, a hostcompound can be a cyclodextrin or cyclodextrin derivative exhibitingwater solubility at 25° C. of greater than 5 mg/mL, such as greater than20 mg/mL, greater than 100 mg/mL, or even greater than 500 mg/mL. Inother particular embodiments, a host compound can be α-amylose,β-amylose or V-amylose. Host compounds can be included at concentrationsranging from about 1 mM to about 1M, for example, from about 5 mM toabout 500 mM, such as from about 5 mM to about 25 nM.

Host compounds can include cyclodextrin derivatives, amylosederivatives, cryptand derivatives, cryptophane derivatives, cavitandderivatives, crown ether derivatives, dendrimer derivatives, nanotubederivatives, calixarene derivatives, valinomycin derivatives, andnigericin derivatives modified with one or more substituents. Forexample, host compounds include amylose derivatives and cyclodextrinderivatives, wherein one or more of the hydroxyl groups or the hydrogenatoms of the hydroxyl groups of their constituent aldose rings arereplaced with substituents. Examples of substituents include acyl groups(such as acetyl groups), alkyl groups, aryl groups, tosyl groups, mesylgroups, amino groups (including primary, secondary, tertiary andquaternary amino groups), halo groups (-F, -Cl, -Br and -I), nitrogroups, phosphorous-containing groups (such as phosphate andalkylphosphate groups), sulfur-containing groups (such as sulfate andsulfate ester groups), bridging groups, (that, for example, connect twoor more hydroxyl positions on a cyclodextrin ring or connect two or morehost compounds), aldehyde groups, ketone groups, oxime groups,carboxylic acid groups and their derivatives, carbonate and carbamategroups, silicon-containing groups, boron-containing groups,tin-containing groups, and hydroxyalkyl groups (such as hydroxyethylgroups and hydroxypropyl groups).

Particular examples of cyclodextrins include α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, and δ-cyclodextrin, and derivatives ofeach of these classes of cyclodextrins. Particular examples ofcyclodextrin derivatives, include hydroxypropylated α-cyclodextrin,hydroxypropylated β-cyclodextrin, hydroxypropylated γ-cyclodextrin,hydroxyethylated α-cyclodextrin, hydroxyethylated β-cyclodextrin,hydroxyethylated γ-cyclodextrin, hydroxyisopropylated α-cyclodextrin,hydroxyisopropylated β-cyclodextrin, hydroxyisopropylatedγ-cyclodextrin, carboxymethylated α-cyclodextrin, carboxymethylatedβ-cyclodextrin, carboxymethylated γ-cyclodextrin, carboxyethylatedα-cyclodextrin, carboxyethylated β-cyclodextrin, carboxyethylatedγ-cyclodextrin, octyl succinated-α-cyclodextrin, octylsuccinated-β-cyclodextrin, octyl succinated-γ-cyclodextrin,acetylated-α-cyclodextrin, acetylated-β-cyclodextrin,acetylated-γ-cyclodextrin, sulfated-α-cyclodextrin,sulfated-β-cyclodextrin and sulfated-γ-cyclodextrin. Other particularexamples of cyclodextrins derivatives include the followingβ-cyclodextrin derivatives: 2,3-dimethyl-6-aminomethyl-β-cyclodextrin,6-Azido-β-cyclodextrin, 6-Bromo-β-cyclodextrin,6A,6B-dibromo-β-cyclodextrin, 6A,6B-diiodo-β-cyclodextrin,6-O-Maltosyl-β-cyclodextrin, 6-Iodo-β-cyclodextrin,6-Tosyl-β-cyclodextrin, Peracetyl-maltosyl-β-cyclodextrin,6-t-butyldimethylsilyl-β-cyclodextrin,2,3-diacetyl-6-butyldimethylsilyl-β-cyclodextrin,2,6-dibutyl-3-acetyl-β-cyclodextrin, 2,6-dibutyl-β-cyclodextrin,2,6-t-butyl-dimethylsilyl-β-cyclodextrin, and2,6-di-O-methyl-3-allyl-β-cyclodextrin. A variety of cyclodextrins andcyclodextrin derivatives can be obtained commercially, for example, fromCTD, Inc. (High Springs, Fla.), or they can be synthesized according toprocedures outlined in the scientific literature, for example, in“Synthesis of Chemically Modified Cyclodextrins,” Croft and Bartsch,Tetrahedron, 39: 1417-1474, 1983.

The term “lower alkanol” refers to a compound having the formula R—OH,where R is an alkyl group having between 1 and 5 carbon atoms such as amethyl group, an ethyl group, an n-propyl group, an isopropyl group, ann-butyl group, a sec-butyl group, a t-butyl group, an n-pentyl group, anisopentyl group or a neopentyl group. Examples of lower alkanols includemethanol, ethanol and isopropanol.

The term “oxidant” refers to an atom or molecule having a greaterreduction potential than a second molecule, for example, a greaterreduction potential than hematoxylin such that it will react with andoxidize hematoxylin to hematein. Oxidants include naturally occurringmolecular oxygen in the atmosphere that diffuses to and oxidizeshematoxylin and a “chemical oxidant” that is actively combined withhematoxylin (typically in solution) to convert at least a portion of thehematoxylin to hematein. Examples of useful chemical oxidants includeone or more of an iodate salt (such as sodium iodate and potassiumiodate), mercuric oxide, a permanganate salt (such as potassiumpermanganate), a periodate salt (such as sodium periodate and potassiumperiodate), and a peroxide (such as hydrogen peroxide). In particularembodiments, the chemical oxidant comprises sodium iodate.

The term “mordant” refers to an ionic metal species with which a dye(such as hematein) can form a complex (such as a cationic complex) thatserves to bind the dye (such as hematein) to particular cellularcomponents such as nuclear DNA, myelin, elastic and collagen fibers,muscle striations and mitochondria. Examples of mordants includealuminum (for example, in the form of an alum such as aluminum sulphate,aluminum potassium sulphate or aluminum ammonium sulphate), iron,tungsten, zirconium, bismuth, molybdenum (phosphomolybdic acid ormolybdic acid), vanadium (vanadate).

The term “water-soluble antioxidant” refers to an antioxidant that has asolubility in water at 25° C. that is sufficient to provide aconcentration of the antioxidant of at least 1 mM, such as greater than5 mM, greater than 10 mM, or even greater than 50 mM.

II. Overview

A stabilized hematoxylin composition is disclosed, which composition canbe used for staining of a biological sample, and in particular, forstaining the nuclei of cells in the biological sample. The compositionincludes mordanted hematein (such as hemalum) stabilized by one or bothof a host compound and an antioxidant. The disclosed hematoxylincompositions show improved stability over similar hematoxylincompositions not including either or both of a host compound and anantioxidant. Likewise, the use of antioxidants and host compounds toincrease stability of other histochemical dye compositions and their usein histochemical staining methods are contemplated. Furthermore, in thecase of hematoxylin, a disclosed hematoxylin composition also appears tohave a higher effective dye concentration that permits darker stainingof biological samples in a predetermined amount of time, which isespecially advantageous in an automated staining method where abiological sample mounted on a microscope slide, and even moreadvantageous if the slide is processed in a horizontal position. Allknown hematoxylin compositions and all histochemical staining methodsutilizing hematoxylin as part of the staining process can benefit fromapplication of the teachings of the present disclosure. Furthermore thebenefits can be extended to “special stains” and the automatedapplication of such special stains to biological samples (such as on theNexES™ Special Stainer (Ventana Medical Systems, Inc., Tucson, Ariz.).

In one embodiment, the disclosed composition includes hematoxylin, asolvent, an amount of a chemical oxidant sufficient to convert at leasta portion of the hematoxylin to hematein, a mordant and either or bothof a host compound and an antioxidant. In particular embodiments, thecomposition includes both a host compound and an antioxidant. In evenmore particular embodiments, the composition includes two or moredifferent antioxidants such as two or more water-soluble antioxidants.In other even more particular embodiments, the composition includes oneor more host compounds and one or more antioxidants.

The host compound of various embodiments can be one or more of anamylose, a cyclodextrin, a cryptand, a cryptophane, a cavitand, a crownether, a dendrimer, a nanotube, a calixarene, a valinomycin, and anigericin. In more particular embodiments, the host compound is one ormore of a cyclodextrin or a cyclodextrin derivative, and moreparticularly one or more of β-cyclodextrin and a β-cyclodextrinderivative. In other more particular embodiments, the host compound canhave a water solubility of greater than 100 mg/mL at 25° C.

In some embodiments, the solvent is an aqueous solvent and theantioxidant is a water-soluble antioxidant. Examples of water solubleantioxidants include hydroquinones; n-alkyl gallates (such as n-propyl,n-octyl, and n-dodecyl gallates); reducible sugars such as sorbitol andmannitol; benzoates and hydroxybenzoates; sulfites and metabisulfites;certain acids such as citric acid, tartaric acid, lactic acid,erythorbic acid ascorbic acid, uric acid, tannic acid, and salts of suchacids (such as Mg²⁺, NH₄ ⁺, Na⁺, K⁺ and Ca²⁺ salts); chelators such asEDTA that remove metals that function as oxidants; and choral hydrate.In particular embodiments, the water soluble antioxidant includes one ormore of hydroquinone and n-propyl gallate.

Various solvents can be utilized for the composition, but typically thesolvent comprises one or more of water, a lower alkanol such as ethanol,and a polyol. In particular embodiments, the solvent comprises anaqueous solvent wherein the aqueous solvent comprises water and apolyol. Particular examples of useful polyols include glycerol, ethyleneglycol, propylene glycol, poly (ethylene glycol), and poly (propyleneglycol). Aqueous solvent compositions typically will comprise 5-45% byvolume of one or more of ethylene glycol and propylene glycol, and moretypically 10-30% by volume of one or more of ethylene glycol andpropylene glycol.

The amount of chemical oxidant utilized in some embodiments of thecomposition can be sufficient to completely (such as substantiallyquantitatively) oxidize the hematoxylin to hematein, or sufficient onlyto partially oxidize the hematoxylin to hematein. In particularembodiments, more than half of the hematoxylin is oxidized to hemateinby the chemical oxidant, and in others, less than half of thehematoxylin is oxidized to hematein by the chemical oxidant. Forexample, between 1% and 50% of the hematoxylin can be oxidized tohematein by the chemical oxidant, but more typically, between about 10%and about 30% of the hematoxylin is oxidized to hematein by the chemicaloxidant. In particular examples, the molar ratio of hematoxylin tooxidant used in the composition is between 6:1 and 1:1. It should beunderstood that although the chemical oxidant is considered part of thecomposition, it is converted to its reduction products upon reactionwith the hematoxylin, which reduction products will remain in thecomposition.

The mordant of the composition can be any mordant such as one or more ofan aluminum mordant, an iron mordant, a bismuth mordant, a coppermordant, a molybdenum mordant, a vanadium mordant, and a zirconiummordant. In some embodiments, the mordant comprises an alum, and in moreparticular embodiments, the mordant comprises aluminum sulphate. Themordant can be present in the composition at a concentration greaterthan the concentration of the hematein in the composition (determinableby refractometry, thin-layer chromatography or spectroscopy), or it canbe present in the composition at a concentration less than theconcentration of the hematein in the composition. Alternatively, in someembodiments, the molar ratio of hematoxylin to mordant in thecomposition is between 2:1 and 1:100, and in particular embodiments, themolar ratio of hematoxylin to mordant in the composition is between 1:5and 1:20.

In some embodiments, the composition further includes an acid such asacetic acid. In other embodiments, no acid is added, and the absence ofthe acid surprisingly still provides a stabilized and effectivehematoxylin composition. In other embodiments, the composition furtherincludes a buffer to control pH, for example, a buffer to control the pHnear a pH between 1 and 4, such as a pH near 2.5.

In some particular embodiments, a disclosed composition comprises amixture of water and ethylene glycol as the solvent, sodium iodate asthe oxidant, aluminum sulphate as the mordant, and β-cyclodextrin or aderivative thereof as the host compound. One or more water solubleantioxidants such as hydroquinone and n-propyl gallate also can beincluded in such particular embodiments. In even more particularembodiments, the mixture of water and ethylene glycol comprises from10-40% by volume ethylene glycol and from 60-90% water.

In another aspect, a method is disclosed for histochemical staining of abiological sample, which method includes contacting the biologicalsample with a disclosed hematoxylin composition and can further includecontacting the sample with a counterstain. In some embodiments,contacting the sample with a counterstain comprises contacting thesample with one or more of eosin Y, orange G, light green SF yellowish,Bismark Brown, fast green FCF, OA-6, EA25, EA36, EA50 and EA65. Theformulas and methods of making such counterstains can be found, forexample, in the StainsFile (an internet resource for histotechnologistsmaintained by Bryan Llewellyn); Kiernan, “Histological and Histochemicalmethods: Theory and Practice,” 3^(rd) Ed. Butterworth Heinemann, Oxford,UK; and in Horobin and Kiernan, “Conn's biological stains: a handbook ofdyes, stains and fluorochromes for us in biology and medicine,” 10^(th)ed., Oxford: BIOS, ISBN 1859960995, 2002. In other embodiments,contacting the sample with the hematoxylin composition comprises aprogressive hematoxylin staining protocol. In yet others, contacting thesample with the hematoxylin composition comprises a regressivehematoxylin staining protocol. The method can be automated, and can beperformed on a biological sample that is supported on a substrate suchas a microscope slide. In particular embodiments, the method is used tostain a tissue section or a cytology sample mounted on a microscopeslide. In particular embodiments further including a counterstainingstep, the method can be an H&E staining method or a PAP staining method,and more particularly an automated H&E or PAP staining method.

In a further aspect, a method is disclosed for making a stabilizedhematoxylin composition for histochemical staining of a biologicalsample. In one embodiment, the method for making the stabilizedhematoxylin solution includes forming a hematein solution, adding amordant to the hematein solution to form a staining solution, and addingeither or both of a host compound and an antioxidant to the stainingsolution to form the stabilized hematoxylin composition. In someembodiments, forming the hematein solution comprises dissolvinghematoxylin in a solvent and adding an amount of a chemical oxidantsufficient to covert at least a portion of the hematoxylin to hematein.In particular embodiments, the solvent used to dissolve the hematoxylincomprises an aqueous composition such as composition including water anda polyol. Useful polyols, as indicated before, include glycerol,ethylene glycol and propylene glycol.

While the principles outlined in this disclosure are applied to variantsof Gill's mordanted hematoxylin in the examples that follow, it shouldbe understood that they can be applied to improve the stability of anymordanted hematoxylin used for histochemical staining of biologicalsamples. In addition to Gill's formulations, particular examples of alummordanted hematoxylin histological stains to which a host compoundand/or an antioxidant can be added to improve stability includeAnderson's, Apathy's, Baker's Bennett's, Bohmer's, Bosma's, Bullard's,Carazzi's, Cole's, Debiden's, de Groot's, Delafield's, Duval's,Ehrlich's, Friedlander's, Gadsdon's, Gage's, Galigher's, Garvey's,Graham's, Hamilton's, Harris', Harris & Power's, Haug's, Homeyold's,Kleinenberg's, Krutsay's, Langeron's, Launoy's, Lee's, Lillie's,Lugol's, McLachlan's, Mallory's, Mann's, Martinotti's, Masson's,Mayer's, Mitchell's, Molnar's, Papamiltiades' , Pusey's, Rawitz',Reddy's, Sass', Schmorl's, Slidders', Unna's, Watson's, and Weigert &Wright's. Particular examples of iron-mordanted hematoxylin stainsinclude Anderson's, Cretin's, Faure's, Goldman's, Hansen's,Heidenhain's, Janssen's, Kefalas', Krajian's, Krutsay's, La Manna's,Lillie's, Lillie & Earle's, Masson's, More & Bassal's, Murray's, Paquin& Goddard's, Regaud's, Rozas', Seidelin's, Thomas', Weigert's, andYasvoyn's. A bismuth-mordanted hematoxylin is Roach & Smith's.Copper-mordanted hematoxylins include Bensley's, Cook's and Faure's. Amolybdenum-mordanted hematoxylin is Held's. Vanadium-mordantedhematoxylins include Hedenhain's, and Smith's. A zirconium-mordantedhematoxylin is McNulty & Smith's. Formulas and methods of making andusing such mordanted hematoxylin solutions can be found, for example, inthe StainsFile (an internet resource for histotechnologists maintainedby Bryan Llewellyn); Kiernan, “Histological and Histochemical methods:Theory and Practice,” 3^(rd) Ed. Butterworth Heinemann, Oxford, UK; andin Horobin and Kiernan, “Conn's biological stains: a handbook of dyes,stains and fluorochromes for us in biology and medicine,” 10^(th) ed.,Oxford: BIOS, ISBN 1859960995, 2002. The contents of the two boundreferences cited immediately above are incorporated by reference herein.

Other histological stains and their methods of use (particularlyautomated methods of use) that can benefit from the stabilizing effectsof one or more of an antioxidant and a host compound include dyes suchas acridine dyes, anthraquinone dyes, arylmethane dyes, azo dyes,diazonium dyes, nitro dyes, phthalocyanine dyes, quinine imine dyes,tetrazolium dyes, thiazole dyes and xanthene dyes. Examples of dyesuseful for histological staining include acetyl yellow, acid black 1,acid blue 22, acid blue 93, acid fuchsin, acid green, acid green 1, acidgreen 5, acid magenta, acid orange 10, acid red 4, acid red 26, acid red29, acid red 44, acid red 51, acid red 66, acid red 73, acid red 87,acid red 91, acid red 92, acid red 94, acid red 101, acid red 103, acidroseine, acid rubin, acid violet 19, acid yellow 1, acid yellow 9, acidyellow 23, acid yellow 24, acid yellow 36, acid yellow 73, acid yellowS, acid yellow T, acridine orange, acriflavine, alcian blue, alcianyellow, alcohol soluble eosin, alizarin, alizarin blue, alizarin blue2RC, alizarin carmine, alizarin cyanin BBS, alizarol cyanin R, alizarinred S, alizarin purpurin, aluminon, amido black 10B, amidonaphthol red,amidoschwarz, aniline blue WS, aniline purple, anthracene blue SWR,anthracene blue SWX, auramine 0, azo-eosin, azocarmine B, azocarmine G,azoeosin G, azoic diazo 5, azoic diazo 48, azophloxine, azovan blue,azure A, azure B, azure C, basic blue 8, basic blue 9, basic blue 12,basic blue 15, basic blue 17, basic blue 20, basic blue 26, basic brown1, basic fuschsin, basic green 4, basic green 5, basic orange 14, basicred 2, basic red 5, basic red 9, basic violet 2, basic violet 4, basicviolet 10, basic violet 14, basic yellow 1, basic yellow 2, Biebrichscarlet, Biebrich scarlet R, Bismarck brown Y, brazilein, brazilin,brilliant crocein, brilliant crystal scarlet 6R, calcium red, carmine,carminic acid carmoisine 6R, Celestine blue B, china blue, chlorantinefast red 5B, cochineal, coelestine blue, Chicago blue 4B, chrome violetCG, chromotrope 2R, chromoxane cyanin R, congo Corinth, Congo red,cotton blue cotton red, croceine scarlet crocein scarlet 3B, croceinscarlet MOO, crocin, crystal ponceau 6R, crystal scarlet, crystalviolet, dahlia, diamond green B, direct blue 14, direct blue 58, directred, direct red 10, direct red 28, direct red 80, direct red 81, directyellow 7, durazol blue 4R, durazol blue 8G, eosin B, eosin bluish,eosin, eosin Y, eosin yellowish, eosinol, Erie garnet B, eriochromecyanin R, erythrosine B ethyl eosin, ethyl green, ethyl violet, Evan'sblue, fast blue B, fast green FCF, fast red B, fast yellow, fast yellowextra, fast yellow G, fat black HB, fluorescein, food green 3, galleon,gallamine blue gallocyanin, gentian violet, hello fast rubin BBL,helvetia blue, Hoffman's violet, hydrazine yellow, imperial red, ingrainblue 1, ingrain yellow 1, INT, Kermes, kermesic acid, kernechtrot, Lac,laccaic acid, Lauth's violet, light green, lissamine fast yellow,lissamine green SF, Luxol fast blue, magenta O, magenta I, magenta II,magenta III, malachite green, Manchester brown, Martius yellow, mauve,mauveine, merbromin, mercurochrome, metanil yellow, methylene azure A,methylene azure B, methylene azure C, methylene blue, methylene green,methyl blue, methyl green, methyl violet, methyl biolet 2B, methylviolet 10B, milling yellow 3G, mordant blue 3, mordant blue 10, mordantblue 14, mordant blue 23, mordant blue 32, mordant blue 45, mordant red3, mordant red 11, mordant violet 25, mordant violet 39, naphthaleneblue black, naphthol blue black, naphthol green B, naphthol yellow S,natural black 1, natural red, natural red 3, natural red 4, natural red8, natural red 16, natural red 24, natural red 25, natural red 28,natural yellow 6, NBT, neutral red, new fuchsin, Niagara blue 3B, nightblue, Nile blue, Nile blue A, Nile blue sulfate, Nile red, nitro BT,nitro blue tetrazolium, nuclear fast red, oil red O, orange G, orcein,pararosanilin, Perkin's violet, phloxine B, picric acid, Ponceau 2R,Ponceau 6R, Ponceau B, Ponceau de Xylidine, Ponceau S, pontamine skyblue 5B, primula, primulin, purpurin, pyronin B, pyronin G, pyronin Y,rhodamine B, rosanilin, rose Bengal, saffron, safranin O, scarlet Rscarlet red, Scharlach R, shellac, sirius red F3B, sirius red 4B, siriussupra blue F3R, solochrome cyanin R, soluble blue, solvent black 3,solvent blue 38, solvent red 23, solvent red 24, solvent red 27, solventred 45, solvent yellow 94, spirit soluble eosin, Sudan III, Sudan IV,Sudan black B, Sudan red BK, sulfur yellow S, Swiss blue, tartrazine,thioflavine S, thioflavine T, thionin, toluidine blue, toluyline red,tropaeolin G, trypaflavine, trypan blue, uranin, Vicoria blue 4R,Victoria blue B, Victoria blue R, Victoria green B, water blue I, watersoluble eosin, woodstain scarlet, Xylidine ponceau, and yellowish eosin,and combinations thereof. Formulas and methods of making and usinghistochemical dye solutions discussed in this paragraph (such as in“special stain” procedures in particular histological contexts, or ascounterstains) can be found, for example, in the StainsFile (an internetresource for histotechnologists maintained by Bryan Llewellyn); Kiernan,“Histological and Histochemical methods: Theory and Practice,” 3^(rd)Ed. Butterworth Heinemann, Oxford, UK; and in Horobin and Kiernan,“Conn's biological stains: a handbook of dyes, stains and fluorochromesfor us in biology and medicine,” 10^(th) ed., Oxford: BIOS, ISBN1859960995, 2002. The contents of the two bound references citedimmediately above are incorporated by reference herein.

III. Examples

Although the method and composition of the disclosure can be applied toany histological staining process (manual or automated) or any slidestaining instrument, the disclosed hematoxylin composition isparticularly useful when incorporated into the automated H&E stainingprocess developed for use in the high volume slide processing systemthat is described in U.S. Patent Application Publication Nos.20040002163 and 20050186114 (both of which applications are incorporatedby reference herein). Briefly, the automated slide processing systemthat is described in the aforementioned applications is a high-volumeslide processing system that shuttles trays holding a plurality ofslides in substantially horizontal positions (to minimizecross-contamination) between workstations that perform various slideprocessing operations on the slides. Fresh reagents can be applied toeach slide during processing, and cross-contamination of slides withreagents can be substantially eliminated because the slides are treatedseparately in spaced-apart fashion in the tray. In one configuration,the system includes a radiant heater, a combinedde-paraffinizer/stainer/solvent exchanger workstation, a convection ovenand a coverslipper. A tray of slides bearing paraffin-embedded tissuesamples can be heated under the radiant heater of the system to spreadthe paraffin in the samples for easier removal and also to adhere thesamples to the slides. The tray can then be transported to themultifunctional de-paraffinizer/stainer/solvent exchanger workstation,where slides can be de-paraffinized, stained, and solvent exchanged. Atray of stained slides that is ready for coverslipping can then beshuttled to the coverslipper of the system where coverslips are added tothe slides. Once the slides are coverslipped, the tray can then betransported to the convection oven to cure the coverslips on the stainedslides. The high volume stainer just described is commercially availablefrom Ventana Medical Systems, Inc, Tucson, Ariz.

While the staining system just described can be configured to performany histological staining process, the system was configured to performa progressive H&E stain using the disclosed hematoxylin compositionsthat are described in detail below. A schematic showing the overallprocess is shown in FIG. 1, which process includes: a baking step toadhere the samples to the slides, a de-paraffinization step to removeparaffin from paraffin-embedded samples, a hematoxylin staining step(that can utilize the disclosed hematoxylin compositions), a bluing stepthat raises the pH and turns the hematoxylin blue to provide bettercontrast with the eosin added downstream, an eosin staining step, adifferentiation step that is used to remove excess eosin and turn theeosin various shades of red to pink, a dehydration step to remove waterfrom the sample using 100% ethanol, a step in which the slides areexposed to an elevated temperature and air flow to remove the ethanol, acoverslipping step in which limonene is dispensed to the sample, and acuring step.

Several hematoxylin compositions were investigated in an effort toprovide a stable composition that also provided for a darker nuclearstain (by virtue of having a higher effective initial hemateinconcentration). Traditionally, solutions that have higher concentrationsof hematein and that as a result can stain nuclei more darkly are madeup and used within a few days because such solutions will form copiousamounts of precipitate. Water-soluble antioxidants (in this example,hydroquinone and n-propyl gallate) were added to a variety ofhematoxylin formulations, singly or in combination, to determine whetherthe antioxidants could stabilize the hematein against oxidativedegradation and precipitation, and β-cyclodextrin was used to determineif addition of a host compound could further slow the natural oxidationof hematein and resulting precipitate formation.

In all instances, the hematoxylin formulations were prepared as follows:

-   -   1) Deionized water and either ethylene glycol (25% by volume;        Sigma-Aldrich, St. Louis, Mo.) or propylene glycol (23% by        volume; Sigma-Aldrich, St. Louis, Mo.) were mixed together to        form a solvent for the composition.    -   2) Hematoxylin dye (Dudley Chemical Corp, Lakewood, N.J.), in        the concentrations indicated in FIGS. 2 and 3, was then added to        the solvent to form a hematoxylin solution.    -   3) Sodium iodate (Sigma-Aldrich, St. Louis, Mo.) was added in        the concentrations indicated in FIGS. 2 and 3 and allowed to        oxidize the hematoxylin to hematein, thereby forming a hematein        solution having an initial molar concentration of hematein        approximately equal to the molar concentration of the        hematoxylin minus the molar concentration of the sodium iodate.    -   4) Aluminum sulphate octadecahydrate (J T Baker, Phillipsburg,        N.J.) was added to the hematein solution in the concentration        indicated in FIGS. 2 and 3 to form a hemalum solution.    -   5) Combinations of hydroquinone, n-propyl gallate and        β-cyclodextrin hydrate (all available from Sigma-Aldrich, St.        Louis, Mo.) were then added in the concentrations indicated in        FIGS. 2 and 3 to form the tested compositions.    -   6) The compositions were placed into separate bag-in-box        containers that are used for on-board storage of reagents in the        automated staining system described above.

No acid was added to the compositions used for these examples.

FIGS. 2 and 3 summarize 8 different compositions and the results ofstability testing at several temperatures based upon observation ofprecipitates in the bag-in-box containers. In all cases, the addition ofone or more antioxidants and the host compound improved stability incomparison to an equivalent “unstabilized” hematoxylin solution withoutan added antioxidant and/or host compound, which unstabilizedhematoxylin exhibits precipitates throughout the container after oneweek at 2-8° C., after 4 weeks at ambient temperature and at 30° C., andafter 2 weeks at 45° C.

Long term stability testing that included use of stored compositions formanual staining of multi-tissue slides also was performed. Two lots ofan aqueous hematoxylin solution including 25% ethylene glycol (v/v), 20mM hematoxylin, 3.3 mM sodium iodate, 20 mM aluminum sulfateoctadecahydrate, 85 mM hydroquinone and 10 mM β-cyclodextrin hydratehaving a pH of about 2.6 were each packed into multiple bag-in-boxcontainers. One container from each lot was left in ambient conditions,one container from each lot was subjected to freeze-thaw cycling, onecontainer from each lot was subjected to 45 degrees C. to ambient shipstress conditions, and one container from each lot was subjected to 2-8degrees C. to ambient ship stress conditions. At monthly intervals, eachof the containers was inspected for the presence of precipitates and analiquot was removed and checked for pH. The aliquot was then used tomanually stain a microscope slide bearing multiple tissue sections(liver, kidney, colon, skin, and one of tonsil, lymph node or spleen).After a total of 13 months of monthly testing, the solutions in all ofthe different containers consistently did not exhibit precipitates, thepH of each of the solutions in the different containers consistentlyremained stable, and the hematoxylin solutions in the differentcontainers consistently provided acceptable nuclear staining of thetissue sections following the manual staining procedure.

It is to be understood that the disclosed invention is not limited tothe particular embodiments illustrated above and that many changes maybe made without departing from the true scope and spirit of theinvention. For example, additional components such as surfactants can beadded to the disclosed compositions, and other dyes, mordanted orotherwise, can be substituted for the hematoxylin. Furthermore, thoseskilled in the art to which the invention pertains will recognize, or beable to ascertain through no more than routine experimentation, manyequivalents to the embodiments described herein. Such equivalents areintended to fall within the scope of the claims.

1.-34. (canceled)
 35. A method for staining a biological sample,comprising: contacting the biological sample with a stabilizedhematoxylin composition, the hematoxylin composition comprising asolvent, hematoxylin, an amount of a chemical oxidant sufficient toconvert at least a portion of the hematoxylin to hematein, a mordant, anantioxidant, and a host compound, wherein the host compound is selectedfrom the group consisting of a polysaccharide, a cryptand, acryptophane, a cavitand, a crown ether, a dendrimer, a nanotube, acalixarene, a valinomycin, a nigericin, and a derivative thereof. 36.The method of claim 35, further comprising contacting the sample with acounterstain.
 37. The method of claim 36, wherein the counterstain isselected from the group consisting of eosin Y, orange G, light green SFyellowish, Bismark Brown, fast green FCF, OA-6, EA25, EA36, EA50 andEA65.
 38. The method of claim 35, wherein contacting the sample with thehematoxylin composition comprises a progressive hematoxylin stainingprotocol.
 39. The method of claim 35, wherein contacting the sample withthe hematoxylin composition comprises a regressive hematoxylin stainingprotocol.
 40. The method of claim 35, wherein the method is automated.41. The method of claim 35, wherein the biological sample is supportedon a substrate.
 42. The method of claim 41, wherein the substratecomprises a microscope slide.
 43. The method of claim 35, wherein thebiological sample comprises a tissue section or a cytology sample. 44.The method of claim 42, wherein the biological sample comprises a tissuesection or a cytology sample.
 45. The method of claim 44, wherein themethod is automated.
 46. The method of claim 35, wherein the methodcomprises an H&E staining method.
 47. The method of claim 35, whereinthe method comprises a PAP staining method. 48.-53. (canceled)
 54. Themethod of claim 35, wherein the host compound has a water solubility ofgreater than 100 mg/mL at 25° C.
 55. The method of claim 35, wherein theantioxidant is selected from the group consisting of a hydroquinones, an-alkyl gallates, a reducible sugar, a benzoate, a hydroxybenzoate; asulfite, a metabisulfite, citric acid, tartaric acid, lactic acid,erythorbic acid ascorbic acid, uric acid, tannic acid, chelators, choralhydrate, derivatives, and salts thereof.
 56. A method for staining abiological sample, comprising: contacting the biological sample with astabilized staining composition, the staining composition comprising asolvent, a histological stain, an antioxidant, and a host compound,wherein the host compound is selected from the group consisting of apolysaccharide, a cryptand, a cryptophane, a cavitand, a crown ether, adendrimer, a nanotube, a calixarene, a valinomycin, a nigericin, and aderivative thereof.
 57. The method of claim 56, wherein the histologicalstains is selected from the group consisting of an acridine dye, ananthraquinone dye, an arylmethane dye, an azo dye, a diazonium dye, anitro dye, a phthalocyanine dye, a quinine imine dye, a tetrazolium dye,a thiazole dye, a xanthene dyes, and mixtures thereof.
 58. The method ofclaim 56, wherein the biological sample comprises a tissue section or acytology sample.
 59. The method of claim 56, wherein the method isautomated.
 60. The method of claim 56, further comprising contacting thesample with a counterstain.